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Characterization of the flagellar motor composed of functional GFP-fusion derivatives of FliG in the Na + -driven polar flagellum of Vibrio alginolyticus

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ABSTRACT

The polar flagellum of Vibrio alginolyticus is driven by sodium ion flux via a stator complex, composed of PomA and PomB, across the cell membrane. The interaction between PomA and the rotor component FliG is believed to generate torque required for flagellar rotation. Previous research reported that a GFP-fused FliG retained function in the Vibrio flagellar motor. In this study, we found that N-terminal or C-terminal fusion of GFP has different effects on both torque generation and the switching frequency of the direction of flagellar motor rotation. We could detect the GFP-fused FliG in the basal-body (rotor) fraction although its association with the basal body was less stable than that of intact FliG. Furthermore, the fusion of GFP to the C-terminus of FliG, which is believed to be directly involved in torque generation, resulted in very slow motility and prohibited the directional change of motor rotation. On the other hand, the fusion of GFP to the N-terminus of FliG conferred almost the same swimming speed as intact FliG. These results are consistent with the premise that the C-terminal domain of FliG is directly involved in torque generation and the GFP fusions are useful to analyze the functions of various domains of FliG.

No MeSH data available.


Fractionation by sucrose density gradient centrifugation. The basal bodies were prepared from MK1 cells harboring plasmids pTY102 (FliG) (a), pTY201(GFP-FliG) (b), or pTY202 (FliG-GFP)(c). The basal bodies (lane 0) were applied to a 20–60% (w/w) stepwise sucrose gradient in TEC. After centrifugation at 72,000×g for 90 min at 4°C, the gradient was divided into 20 fractions from the top to the bottom. Proteins in each fraction were separated by SDS-PAGE and were detected by immunoblotting using anti-MotY (upper panel) or anti-FliG antibodies (lower panel).
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f5-7_59: Fractionation by sucrose density gradient centrifugation. The basal bodies were prepared from MK1 cells harboring plasmids pTY102 (FliG) (a), pTY201(GFP-FliG) (b), or pTY202 (FliG-GFP)(c). The basal bodies (lane 0) were applied to a 20–60% (w/w) stepwise sucrose gradient in TEC. After centrifugation at 72,000×g for 90 min at 4°C, the gradient was divided into 20 fractions from the top to the bottom. Proteins in each fraction were separated by SDS-PAGE and were detected by immunoblotting using anti-MotY (upper panel) or anti-FliG antibodies (lower panel).

Mentions: The GFP-fused FliG is assembled into the flagellar motor and is functional. We wanted to see the GFP-fused proteins directly by electron microscopy in the flagellar rotors. Rotors (basal bodies) with GFP-fused FliG were isolated using a protocol developed to isolate the basal bodies of V. alginolyticus with FliG38. The basal bodies with GFP-FliG or FliG-GFP were isolated and fractionated by sucrose density gradient centrifugation. MotY and GFP-fused FliG were detected by western blotting (Fig. 5). The band peaks of both GFP-fused FliG proteins copurified with MotY, which is a marker protein of the basal body. We confirmed that the GFP-fused FliG proteins are associated with the basal body similar to wild-type FliG, as previously reported38. FliG was also detected in the upper fractions of the gradient (1 to 5), indicating that FliG is easily dissociated from the basal body. More FliG was detected in the upper fractions of the gradient from the GFP fused FliG than from the intact FliG. As for FliG-GFP, the degradation bands were detected in the upper fractions. The basal bodies from peak fractions of FliG were observed by electron microscopy with negative staining. However, no structural difference due to the fused GFP was observed (data not shown). We may have to find a milder condition to purify the basal body or to make a stable construct of the fusion, such as the linker sequence is optimized.


Characterization of the flagellar motor composed of functional GFP-fusion derivatives of FliG in the Na + -driven polar flagellum of Vibrio alginolyticus
Fractionation by sucrose density gradient centrifugation. The basal bodies were prepared from MK1 cells harboring plasmids pTY102 (FliG) (a), pTY201(GFP-FliG) (b), or pTY202 (FliG-GFP)(c). The basal bodies (lane 0) were applied to a 20–60% (w/w) stepwise sucrose gradient in TEC. After centrifugation at 72,000×g for 90 min at 4°C, the gradient was divided into 20 fractions from the top to the bottom. Proteins in each fraction were separated by SDS-PAGE and were detected by immunoblotting using anti-MotY (upper panel) or anti-FliG antibodies (lower panel).
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Related In: Results  -  Collection

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f5-7_59: Fractionation by sucrose density gradient centrifugation. The basal bodies were prepared from MK1 cells harboring plasmids pTY102 (FliG) (a), pTY201(GFP-FliG) (b), or pTY202 (FliG-GFP)(c). The basal bodies (lane 0) were applied to a 20–60% (w/w) stepwise sucrose gradient in TEC. After centrifugation at 72,000×g for 90 min at 4°C, the gradient was divided into 20 fractions from the top to the bottom. Proteins in each fraction were separated by SDS-PAGE and were detected by immunoblotting using anti-MotY (upper panel) or anti-FliG antibodies (lower panel).
Mentions: The GFP-fused FliG is assembled into the flagellar motor and is functional. We wanted to see the GFP-fused proteins directly by electron microscopy in the flagellar rotors. Rotors (basal bodies) with GFP-fused FliG were isolated using a protocol developed to isolate the basal bodies of V. alginolyticus with FliG38. The basal bodies with GFP-FliG or FliG-GFP were isolated and fractionated by sucrose density gradient centrifugation. MotY and GFP-fused FliG were detected by western blotting (Fig. 5). The band peaks of both GFP-fused FliG proteins copurified with MotY, which is a marker protein of the basal body. We confirmed that the GFP-fused FliG proteins are associated with the basal body similar to wild-type FliG, as previously reported38. FliG was also detected in the upper fractions of the gradient (1 to 5), indicating that FliG is easily dissociated from the basal body. More FliG was detected in the upper fractions of the gradient from the GFP fused FliG than from the intact FliG. As for FliG-GFP, the degradation bands were detected in the upper fractions. The basal bodies from peak fractions of FliG were observed by electron microscopy with negative staining. However, no structural difference due to the fused GFP was observed (data not shown). We may have to find a milder condition to purify the basal body or to make a stable construct of the fusion, such as the linker sequence is optimized.

View Article: PubMed Central - PubMed

ABSTRACT

The polar flagellum of Vibrio alginolyticus is driven by sodium ion flux via a stator complex, composed of PomA and PomB, across the cell membrane. The interaction between PomA and the rotor component FliG is believed to generate torque required for flagellar rotation. Previous research reported that a GFP-fused FliG retained function in the Vibrio flagellar motor. In this study, we found that N-terminal or C-terminal fusion of GFP has different effects on both torque generation and the switching frequency of the direction of flagellar motor rotation. We could detect the GFP-fused FliG in the basal-body (rotor) fraction although its association with the basal body was less stable than that of intact FliG. Furthermore, the fusion of GFP to the C-terminus of FliG, which is believed to be directly involved in torque generation, resulted in very slow motility and prohibited the directional change of motor rotation. On the other hand, the fusion of GFP to the N-terminus of FliG conferred almost the same swimming speed as intact FliG. These results are consistent with the premise that the C-terminal domain of FliG is directly involved in torque generation and the GFP fusions are useful to analyze the functions of various domains of FliG.

No MeSH data available.